Capillary electrophoresis has achieved a remarkably rapid development from its introduction in the early 1980s. This technique miniaturizes the electrophoretic process and presents remarkable advantages over traditional slab gel electrophoretic techniques. The most significant limitation in slab gel electrophoresis is the Joule heating which results from current flow through the system. When the electrophoretic process is carried out in a capillary with an internal diameter of 25-100 .mu.m, the Joule heat is dissipated from the surface more efficiently than from any other support, allowing the use of higher voltage and increasing separation efficiency and velocity.
Separation efficiency of columns in capillary electrophoresis can be expressed as a number of theoretical plates generated per a unit length of column for a standard substance. The equation relating number of theoretical plates N to mobility of analyte .mu., voltage V, and diffusion coefficient D is: EQU N=.mu.V/2D.
This equation takes into account only diffusion as a factor influencing separation efficiency. Nevertheless, there are other effects which influence separation efficiency: electromigration dispersion, produced Joule heat, the length of the sampling plug, sorptions on the capillary wall, length of detection cell, and eddy migration. Sorption and eddy migration are directly affected by the quality of the inner capillary surface. A fused silica capillary has approximately 5 silanol groups per square nanometer. Silanol groups behave as a weak acid, ionizing in water with a broad titration curve from pH 2 to 9. Silanols' dissociation generates charges as an integral part of the wall. If an analyte interacts with the capillary wall, typically by Coulombic interactions, the migration of molecules close to the capillary wall is decelerated and the analyte peak is tailing.
Distribution of macromolecules between the buffer and the wall causes bandspreading and poor reproducibility of transit times. Reversible interactions between analytes and a capillary surface worsen the separation profile, broadening the peaks and decreasing reproducibility, while irreversible interactions completely destroy the profile.
Presence of a negative charge on the capillary surface is a source of electroosmotic flow: the negatively charged surface is supposed to migrate in the electric field; because the charged groups are immobilized on the capillary surface, it is the liquid inside the capillary which moves, generating so-called electroosmotic flow. In some respect the presence of electroosmotic flow can be advantageous, since it allows analytes with very low mobilities to get through the detector (e.g., gamma globulins of serum proteins, or simultaneous analysis of cations and anions as frequently needed in the case of peptide mapping). The electroosmotic flow profile is typically of a plug-shape and does not deteriorate separation significantly. However, if the surface charge at the capillary surface is not homogenous, an electroosmotic flow of different velocity generates lengthwise along the capillary which generates eddy migration (see FIG. 1). Eddy migration causes significant peak broadening and loss of separation efficiency. Therefore, to achieve high separation efficiencies, the electroosmotic flow has to be equalized. However, if there is a residual charge on the capillary surface, this allows sorption on the wall and thus locally reduces the electroosmotic flow. Generated inhomogeneities cause eddy migration. Because of that, electroosmotic flow needs to be eliminated completely to achieve the highest separation efficiency.
There are typically two approaches to the elimination of the electroosmotic flow. In the first approach, a dynamic coating is formed. This can be effected by addition of a polymer or a surfactant to the background electrolyte (BGE) or is made by adding suitable cations (polycations, cationic detergents, multicharged cations). The neutral polymer interacts with the capillary wall and shields it from the liquid. Simultaneously, it increases viscosity in the electric double layer and thus reduces electroosmotic flow. Various cellulose derivatives and other hydrophilic polymers have been used for years for this purpose. Examples of this technique include use of polyethyleneoxide, described by Iki and Yeung (J. Chromatogr. A 731, 1996, 273-282) and the use of a copolymer of hydroxypropylcellulose and hydroxyethyl-methacrylate described by Huang, et al. (Electrophoresis 16, 1995, 396-401).
Cationic additives titrate the negative charge of the capillary wall so that the electrokinetic potential and electroosmotic flow are decreased, neutralized or even reversed. In some cases the capillary is flushed with a solution of the cation prior to filling with BGE instead of adding the cation to BGE. Polycations and cationic surfactants have been used recently to coat the capillary wall dynamically, as described by Cifuentes, et al. (J. Chromatography B 681, 1996, 21-27).
Better results are obtained if a permanent (or static) wall coating is formed. Permanent wall coatings for capillary electrophoresis usually consists of two layers. An early permanent coating was described by Hjerten in J. Chromatography 347, 1985, 191-198. Hjerten describes attaching bifunctional 3-methacryloxypropyltrimethoxysilane to the capillary wall in the presence of linear polyacrylamide. In this way, electroosmosis and sorptions were eliminated. However, 3-methacryloxypropyltrimethoxysilane is attached to the capillary wall via siloxane bonding which is hydrolytically unstable at alkali pH. That is why a capillary coated with this film is stable at pH.ltoreq.7 only. Given that many electrophoretic protocols require alkali pH, an alkali stable coating is needed.
Novotny et al. (U.S. Pat. No. 5,143,753) describe a protective coating from polyacrylamide, using vinylmagnesium bromide to attach the polymer to the capillary wall. In the beginning of the procedure, the fused silica surface is activated by reaction with thionyl chloride and the vinyl group is connected by the reaction with vinylmagnesium bromide. The formed coating is attached to the capillary wall via Si--C bond and not via siloxane bond; therefore, a significantly better stability of the coating is achieved at alkali pH. The capillary is free of any electroosmotic flow and the coating is stable at pH=9.5 for approximately 7 days; after that the migration times get longer, which is a result of increasing electroosmotic flow. This is a consequence of hydrolysis of amide groups in polyacrylamide and an increasing electrokinetic potential on the capillary wall. The polymer shields the residual silanol groups and simultaneously increases viscosity in the electric double layer.
Besides .gamma.-methacryloxypropyltrimethoxysilane and Grignard chemistry, bifunctional reagents have also been used to attach a polymer layer to the capillary wall. Examples include Huang, et al. (J. Microcol. Sep. 4, 1992, 135-143) using bifunctional crosslinked siloxane, and chiari, et al. (J. Chromatogr. A 717, 1995, 1-13) using catalytical hydrosilylation followed by reaction with allylmethacrylate.
Polyacrylamide is the most frequently used polymer for wall coating. However, its hydrolytic instability limits the life time of the prepared wall coating. Crosslinking polyacrylamide is one way to increase the stability of the coating. Another way to increase the stability of the wall coating is either to replace acrylamide with a more stable monomer, such as acryloylaminoethoxyethanol as described by Chiari, et al. (J. Chromatogr. A 717, 1995, 1-13; see also Righetti & Chiari, U.S. Pat. No. 5,470,916), acryloylamino-ethoxyethylglucose as described by Chiari et al. (Anal Chem 68, 1996, 2731-2736), or acryloylaminopropanol as described by Simo-Alfonso, et al., Electrophoresis 17, 1996, 723-731), or to use a chemically activated stable polymer, such as hydroxymethylcellulose or dextran as described by Hjerten and Kubo (Electrophoresis 14, 1993, 390-395). Acryloylaminoethoxyethylglucose provides a hydrolytically very stable coating which, at pH 8.5, supports without an obvious electroosmotic flow and a loss of resolution at least 300 runs for separation of proteins.
As the above shows, there are many possibilities to limit electroendoosmosis in capillary tubes. However each has limitations.
It is the initial object of the invention to describe a novel compound for minimizing the effects of electroosmosis and a method to produce this compound. The monomers of this compound must be able to be polymerised into a stable polymer.
It is another object of the invention to produce a coating for the interior of capillary tubes that reduces electrooosmosis.
It is a further goal to produce a coating that is stable at a wide pH range including alkaline pH levels.
It is a further goal to produce a coating that is stable for hundreds of electrophoretic runs. Once a capillary coating degrades, it has to be replaced. This produces material costs. Any coating that allows additional electrophoretic runs without degradation would help minimize these costs.